Molecular genetic changes that underlie the initiation of breast cancer are poorly defined, and the identification of such events is a major focus of breast cancer research. In order to identify these changes we compared the gene expression profiles of normal mammary epithelial cells, and in situ (DCIS ductal carcinoma in-situ), invasive and metastatic breast carcinomas using SAGE (Serial Analysis of Gene Expression). Through the pair-wise comparison of these SAGE libraries, we have identified a novel gene HIN-1 (High In Normal-1) that is present only in normal luminal mammary epithelial cells. Subsequently we have shown that HIN-1 expression is significantly down regulated in 95% of human breast carcinoma specimens including early stage breast carcinomas such as ductal and lobular carcinoma in-situ. The expression of HIN-l is silenced by methylation in the majority of breast cancer cell lines (>95%) and primary tumors (>70%). HIN-1 appears to be a novel secreted protein with no homology to known proteins based on predicted amino acid sequence. A putative high affinity HIN-1 receptor is present in normal and cancerous mammary epithelial cells suggesting an autocrine mechanism of action. Reintroduction of HIN-1 into breast cancer cells appears to inhibit cell growth and the expression of HIN-1 is restricted to terminally differentiated epithelial cells. Based on these recently published and preliminary data, we hypothesize that HIN-1 is a breast tumor suppressor gene that is an autocrine epithelial growth factor. The current proposal is aimed at the further characterization of the function of the HIN-l gene. The specific aims of this proposal are (1) to test the hypothesis that HIN-l is an autocrine factor that regulates mammary cell growth, morphogenesis, and survival, (2) to analyze the in vivo expression of HIN-1 in developing and adult normal and cancerous breast tissue, and (3) to test the hypotheses that HIN-1 is required for mouse mammary gland development and loss of HIN-1 is sufficient for mouse mammary tumorigenesis. The functional characterization of BIN-1 and the signaling pathways in which it is involved will not only further our understanding of the molecular basis of mammary development and tumorigenesis, but will also provide new and valuable targets for future translational research.